Derivatives of ovarian follicular hormones and process of obtaining same



Patented Jan. 26, 1937 v 2,069,096 PATENT OFFICE DERIVATIVES OF OVARIANFOLLICULAR HORMONES AND PROCESS OF OBTAIN- ING SAME Donald W.MacCorquodale and Sidney A. Thayer, St. Louis, and Edward A. Doisy,Webster Groves, Mo., assignors to President and Board of Trustees of St.Louis University, St. Louis, M0.

N0 Drawing.

Application August 9, 1933, Serial 5 Claims.

The invention relates to certain new chemical substances and moreparticularly to oxidation products of the beta ovarian hormone and itssimple chemical equivalents. In general, the new substances of ourinvention have estrogenic activity;

Our invention is based on the discovery that substances can be obtainedby oxidizing under controlled conditions the ovarian follicular hormonesobtained from natural sources. We have discovered that it is desirableto protect the phenolic group from oxidation either by selection of anoxidation method which will act pref erentiallyon other portions of themolecule or by chemically treating said phenolic group to convert thesame to a form more resistant to oxidation. Thus the beta ovarianhormone,

C1BH24O3, may be treated by fusion with potassium hydroxide inorder toobtain an oxidized compound or it may be first converted into the methylether derivative and then oxidized with permanganate to obtain anotheroxidation product which in turn may be reconverted into a compoundhaving the phenolic hydroxyl group.

Our invention contemplates a large class of compounds which may bedescribed as the oxidation products of the ovarian follicular hormonesand/or such derivatives as have the phenolic hydroxyl group protected,which oxidation products have at least 2 of the 4 rings of the originalcompound intact. Examples of various compounds representative of theclass are hereinafter given.

Example 1.-Meth0d-of preparing phenolic dibasic acid, C18H2205, from thebeta ovarian follicular hormone, CiaHmOs.

500 mg. of the beta ovarian follicular hormone were stirred into a meltof 10 gm. of potassium hydroxide and 1.5 cc. of water in a nickelcrucible held at 275 in a bath of fused sodium and potassium nitrates.Heating at this temperature was. continued for 1 hour with occasionalstirring. After the melt had cooled it was dissolved in water andfiltered from a small amount of nickel oxide. Saturation of the solutionwith carbon dioxide resulted in the precipitation of about 50 mg. ofbeta hormone which was removed by filtration. The filtrate was acidifiedwith sulfuric acid, cooled thoroughly, and the precipitated acidfiltered oli, washed with water and dissolved in alcohol. The lightbrownsolution was treated with norlt and the acid subsequently crystallizedfrom dilute alcohol. A very effective means of purification was found inthe precipitation of the potassium salt from absolute alcohol. Thematerial was dissolved in absolute alcohol and treated with a solutionof potassium hydroxide in absolute alcohol. The granular white potassiumsalt was filtered ofi, washed with absolute alcohol, dissolved inwacontained 1 molecule of water of hydration which was removed byheating in a vacuum at over phosphorus pentoxide, or by standing oversulfuric acid. The anhydrous acid melted at 210-21l.

C18H2205 Calculated C 67.88, H 6.97, mol. Wt.

318 C 67.35, 67.55, H 7.03, 6.88, mol. wt. 318, 320 CmHzzOo-HzOCalculated H2O 5.36

Found E20 4.73, 4.88

Found The new compound probably has the condensed structural formula,HOC16H19(CO2H)2. I

Another method for producing the same compound, C1sH22O5, is to firstalkylate the beta hormone thereby obtaining an alkyl ether of the betahormone. This compound is then oxidized by permanganate in a mannerhereinafter more fully set forth and is subsequently hydrolyzed byfusion with caustic potash or other suitable methods commonly used forhydrolysis of ethers.

Example 2.-Method used in preparing an oxidized product, C1'1H220s, fromthe alpha ovarian follicular hormone, CmHzaOz.

'100 mg. of the alpha ovarian follicular hormone, C1aH22O2, were fusedwith 5.0 gm. of potassium hydroxide and 0.75 cc. of water in a nickelcrucible at 275 for 1% hours. The brown fusion was dissolved in waterand filtered, the

filtrate saturated with carbon dioxide, and the C17H22O3 Calculated C74.40, H 8.09

" Found C 74.49, 74.40, 74.24,

The acid readily reddens litmus paper when in dilute alcoholic solution.Its phenolic character is indicated by the formation of an orange-reddye when its alkaline solution is treated with diazotized sulfanilicacid.

Example 3.--Method used in preparing an oxidation product, CwHz-lOs,designated as the methyl ether of the above phenolic dibasic abid.

'15 mg. of acid, Ciel-1220s, (Example 1) were dissolved in 15 cc. ofmethyl alcohol and 2 cc. of redistilled dimethyl sulfate added.

15 cc. of 25% potassium hydroxide were added and the mixture was heatedin an oil bath at 110, under a reflux condenser for hour. Then 1 cc. ofdimethyl sulfate and cc. of potassium hydroxide were addedand theheating was continued for 2 hours. A small amount of ester separated asan oil and this was saponifled by further heating with additional alkaliThe cold solution was acidified with hydrochloric acid forming a whiteprecipitate. This was filtered oljf, dissolved in methyl alcohol, andtreated with norit to remove a small amount of color. After tworecrystallizations from dilute methyl alcohol the yield of whitecrystalline product was 538 mg. Further recrystallization from the samesolvent gave a product which melted CioHaOs: Calculated C 68.63, H 7.28,01130 9.33

' portions as the oxidation proceeded. At first the mol, wt. 332

C 68.49, 68.59, H 7.1, 7.1, CHsO 9.68, 9.70, 9.33. mol. from CHaO value,324

Example 4. Another method used in preparing CiaHaOs.

' Another method for preparing the methyl Found ether of the phenolicdibasic acid is to convert the reaction was slow but the speed ofreaction increased after the oxidation was well started.

When permanganate equivalent to 3 atoms of available oxygen had beenconsumed, the reaction slackened perceptibly and after 0.55 gm.

,additional had been added, the reaction stopped.

After standing for 24 hours the supernatant liquid was colorless. It wasseparated by filtration and the residue washed with dry acetone.

The residue was leached thoroughly with normal KOH solution and thenwith water. Acidification of these combined washings with dilutesulfuric acid precipitated a brown gummy mass which gradually hardenedon standing. The precipitate was washed thoroughly with water and dried.The weight was about 4 gm. It was dissolved in absolute alcohol andtreated with an absolute alcoholic solution of potassium hydroxide.

A light-brown granular salt separated from the dark-brown solution. Itwas filtered oil, the filtrate being reserved for further purificationas hereinafter described. The light-brown granular salt was washed withabsolute alcohol and then with dry ether. It was then dissolved in waterand the solution acidified with hydrochloric acid,

whereupon a brown gummy mass separated which gradually hardened onstanding. This was washed thoroughly with water and was then dissolvedin methyl alcohol and treated with norit. The filtrate, which stillcontained some color. was evaporated to dryness and the residuedissolved in hot glacial acetic acid. About 2 01- f wt., calculated 7umes of water were added to the hot solution, which on cooling depositeda. brown crystalline solid. This was dissolved 'in methyl alcohol anddecolorizedwith norit. Upon dilution with water and evaporation of someof the alcohol the solu- 5 tion deposited white crystals which whendried had a melting point of 192 (uncorrected) The product is the sameas produced in Example 3 and has the formula, CisH2405., The yield was1.79 gm.

Example 5.-A method used in preparing CraHaaOa from the filtrate ofExample 4.

The reserved alcoholicfiltrate from the pre-, cipitated potassiumsaltwas diluted with water and heated on the water bath to removealcohol. Upon acidification with hydrochloric acid a brown gummy massprecipitated. This was washed, dried and treated with methyl alcohol.whereupon most of the material dissolved, leaving a difiicultly solublecrystalline mass. This material was washed with cold methyl alcohol andthen dissolved in about 25 cc. of hot ethyl alcohol and decolorized withnorit. The filtrate was concentrated to 4 cc. on the water bath, and oncooling deposited sheaves of long, well formed, needle-like plates. Theyield was 1'75 mg. and the melting point 182. I

018K220: Calculated c 75.41735, cmo

Example .6.-Method used in preparing CrsHnOs from the product,019112405, of Example 3.

572 mg. of the acid, C19H2405 (melting point Found 30 194, uncorrected),were dissolved with warming in a solution of 500 mg. of potassiumcarbonate in 50 cc. of water. At room temperature this solution wascautiously treated with a 0.1 N potassium permanganate solution, atfirst drop-bydrop with shaking, and then 10 cc. at a time, each timeafter the previous portion had been completely reduced. Toward the endthe reaction slackened and the permanganate was added more rapidly. Inall, 410 cc. were added. After standing at room temperature for 2 hoursthe solution contained only a trace of permanganate and this was reducedby warming on the water bath. The manganese dioxide was removed byfiltration and the filtrate concentrated on the water bath to a smallvolume and then acidified with dilute sulfuric acid. The brown resinousprecipitate was dissolved in ethyl alcohol and treated withhorit but itcould not be induced to crystallize. It was then dissolved in absolutealcohol and treated with an absolute alcohol solution of potassiumhydroxide. The brown potassium salt thus obtained was dissolved in waterand theacid precipitated by means of dilute hydrochloric acid. Repeatedcrystallization of this from dilute alcohol finally yielded a smallamount of white crystalline acid which melted at 234-235".

Ciel-1220s Calculated C 69.65, H 6.72. CHaO 9.4

Found C 69.19, 68.60, H 6.6, 6.8,

- CHaO 9.5 9.5. 0.0101 gm. required 1.21 cc. of alkali (neutralizingiactor=0.04885) Neutralization equivalent calculated for the dibasicacid, CnHaaOa, was 165; found, 167.

Example 7.-Method used in preparing another oxidation product. CiaHuiOs,from CmHaaOs.

By means of a diiierent oxidation process wethe mixture warmed on thewater bath. The to-- tal volume of oxidizing solution used was 41cc. Themixture was warmed on the water bath until the chromate was completelyreduced and it was then diluted with about 4 volumes of water and set inthe ice box to cool. The light-brown granular precipitate whichseparated was filtered ofi, dissolved in '75 cc. of 95% alcohol, andtreated with norit. The filtered solution was concen traded to 10 cc.and on cooling deposited fine White needles. The yield was '75 mg. andthe melting point 243-244". 4

C18Hl805 Calculated C 68.76 H 5.77, CHBO 9.87

Found C 68.94, 68.86, H 5.88, 5.99,

CHsO 9.60 9.90.

The substance is neutral to litmus and insoluble in cold alkali but onprolonged heating it passes slowly into solution. It does not react withsemicarbazide or with hydroxylamine. The neutralization equivalent wasdetermined byhe'ating the substance with an excess of standard alkaliand titrating the excess base with acid. The value obtained, 175,indicates that the substance is the dilactone of a dihydroxy dibasicacid. The calculated value for the neutralization equivalent is 157.

All of the products previously described are crystalline substances inwhich the phenolic hy droxyl group has not been oxidized although theproducts all represent oxidation degradation compounds compared with thenatural ovarian hormones. It is characteristic of the new series ofcompounds that at least 2 of the 4 rings of the ovarian hormone arestill intact. While the compounds specifically described have usuallybeen in the form of the acid substance, it is to be understood that thecorresponding metal salts can readily be prepared in the usual mannerand our invention contemplates the alkali, alkaline earth metal andheavy metal salts as well. general, the sodium salts are desirable formaking solutions for therapeutic purposes. However, other salts and theacid substances themselves or their derivatives such as esters andamides may be used; in fact, any derivatives which readily split-downinto these carboxylic acids may be administered without departing fromthe spirit of this invention. While we have indicated that our inventioncontemplates the protection of the phenolic hydroxyl group bymethylation, it is to" be understood that there are many equivalentmethods of obtaining the same results. For example, the methods ofesterification or etherification may be used. Any alkyl compound may besubstituted for the methyl compound such as the ethyl and propylderivatives. Furthermore, acids such as acetic or benzoic acid may beused to form the acetyl or benzoyl compounds prior to the oxidationstep. Thus the process outlined above will obviously suggest many otherequivalents, all of which come within the purview of the in- 'vention.

It is also understood that in lieu of the oxide.-

tion by permanganate and chromic acid, other oxidation methods may beused which will not break-down the protected phenolic portion of themolecule. Also, other less violent oxidation methods may be employedwhen compounds are treated which do not have the phenolic hydroxylprotected.

The complete structural formula of the ovarian hormones and the class ofnew derivatives herein described and claimed is not definitely known. Itis believed, however, that the beta hormone, C1sH24O3, may be designatedas /CHOH HOClaHn CHOH correspondingly the formula of the acid compound,C1aH2205, of Example 1 is probably COOH HOCmHm The alpha hormone,C18H22O2, has the condensed formula cri, HOCmH19\ The compound of Exmple2, C1'1H22O3, has the condensed structural formula HO CicH20-C O OH Thecompound of Examples 3 and 4, C19H24O5, has formula coon CHaOCmHn coonExample 5, C18H2203, is

COOH CHsO Example 6, C19H22O5, is

What we claim as our invention is: 1. The phenol type compounds where Xis H or a radical capable of protecting the nal unoxidized substance;and wherein the phenolic ring is unoxidized, being solids of deflnitemelting points when in the pure state, pos- 15 sessing at least onecarhoxylic acid group and not less than fifteen carbon atoms in the freephenol part of the compounds.

5. The process comprising reacting a'member of the group consisting ofan ovarian follicular hormone and its simple chemical equivalent havingthe phenolic CH of the hormone protected against oxidation, with anoxidizing'agent capable of oxidizing the hormone substance to a.carboxylic acid containing at least two of the four rings of theoriginal hormone, including the phenolic ring. i Y DONALD W;MACCORQUODAIE.

SIDNEY A. THAYER.

EDWARD A. DOISY.

